The overall objective is to characterize the interrelationships between Ca2 ion and cAMP in prolactin synthesis and secretion. The GH3 cloned strain of rat pituitary tumor cells, which synthesize and release prolactin and which exhibit responses of typical normal pituitary tissue, will be employed as a model system. Ca2 ion and cAMP are reported to influence prolactin synthesis and release in these cells. Furthermore we have obtained evidence that high concentrations of multifunctional Ca2 ion - dependent regulator protein (CDR) and a Ca2 ion - dependent form of adenylate cyclase thought to be restricted in tissue and species distribution are present in GH3 cells. Therefore, (1) the Ca2 ion - dependent adenylate cyclase will be characterized with respect to dependency on CDR and to regulation by GTP and hormones recognized to influence pituitary function. The abilities of Ca2 ion to modulate accumulation of cAMP and of hormones to regulate free Ca2 ion concentrations will be examined in intact cells. The role of CDR in the development of subsensitivity to TRH will be assessed. (2) The importance of Ca2 ion, cAMP CDR and protein kinase and of interactions between these substances to the synthesis of prolactin in intact cells will be determined. Evidence for specific Ca2 ion - dependent and cAMP-dependent protein phosphorylations will be sought using two-dimensional gel electrophoretic techniques. (3) The possibility that Ca2 ion.CDR is an obligatory intermediate in the process of prolactin secretion will be investigated. (4) Prolactin synthesis and release in GH3 cells and in normal pituitary cells will be compared.